CN103756930A - Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof - Google Patents
Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof Download PDFInfo
- Publication number
- CN103756930A CN103756930A CN201310643869.3A CN201310643869A CN103756930A CN 103756930 A CN103756930 A CN 103756930A CN 201310643869 A CN201310643869 A CN 201310643869A CN 103756930 A CN103756930 A CN 103756930A
- Authority
- CN
- China
- Prior art keywords
- peanut
- strain
- fungi
- bacterium
- bacterial strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000017060 Arachis glabrata Nutrition 0.000 title claims abstract description 72
- 235000010777 Arachis hypogaea Nutrition 0.000 title claims abstract description 72
- 235000018262 Arachis monticola Nutrition 0.000 title claims abstract description 72
- 235000020232 peanut Nutrition 0.000 title claims abstract description 72
- 241000894006 Bacteria Species 0.000 title claims abstract description 45
- 230000000443 biocontrol Effects 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 241001553178 Arachis glabrata Species 0.000 title abstract 7
- 238000012360 testing method Methods 0.000 claims abstract description 29
- 239000002689 soil Substances 0.000 claims abstract description 27
- 241000233866 Fungi Species 0.000 claims abstract description 22
- 244000105624 Arachis hypogaea Species 0.000 claims description 65
- 230000001580 bacterial effect Effects 0.000 claims description 46
- 244000005700 microbiome Species 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 23
- 238000002474 experimental method Methods 0.000 claims description 16
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- 235000009973 maize Nutrition 0.000 claims description 14
- 241001157813 Cercospora Species 0.000 claims description 11
- 230000000452 restraining effect Effects 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims description 3
- 235000011613 Pinus brutia Nutrition 0.000 claims description 3
- 241000018646 Pinus brutia Species 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 3
- 229960001225 rifampicin Drugs 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 241000193417 Brevibacillus laterosporus Species 0.000 abstract description 26
- 230000000694 effects Effects 0.000 abstract description 26
- 201000010099 disease Diseases 0.000 abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 16
- 206010039509 Scab Diseases 0.000 abstract description 15
- 230000003042 antagnostic effect Effects 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 abstract 4
- 239000004471 Glycine Substances 0.000 abstract 2
- 241000519856 Phyllosticta Species 0.000 abstract 2
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 244000052769 pathogen Species 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 21
- 238000001179 sorption measurement Methods 0.000 description 13
- 230000002265 prevention Effects 0.000 description 12
- 241000193830 Bacillus <bacterium> Species 0.000 description 11
- 238000000855 fermentation Methods 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 241000726221 Gemma Species 0.000 description 10
- 244000052616 bacterial pathogen Species 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000002538 fungal effect Effects 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 108010022172 Chitinases Proteins 0.000 description 5
- 102000012286 Chitinases Human genes 0.000 description 5
- 206010010254 Concussion Diseases 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000009514 concussion Effects 0.000 description 5
- 230000009977 dual effect Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000011835 investigation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 239000002699 waste material Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241001530056 Athelia rolfsii Species 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 241000555281 Brevibacillus Species 0.000 description 3
- 102000016938 Catalase Human genes 0.000 description 3
- 108010053835 Catalase Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 238000003794 Gram staining Methods 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- TWFZGCMQGLPBSX-UHFFFAOYSA-N carbendazim Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 230000003628 erosive effect Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000012882 sequential analysis Methods 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000223221 Fusarium oxysporum Species 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 244000144992 flock Species 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000009335 monocropping Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241001112741 Bacillaceae Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920000832 Cutin Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000186541 Desulfotomaculum Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 241000223195 Fusarium graminearum Species 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000726219 Gemma gemma Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241001115351 Physalospora Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000813090 Rhizoctonia solani Species 0.000 description 1
- 241000235546 Rhizopus stolonifer Species 0.000 description 1
- 241000192023 Sarcina Species 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 241000204117 Sporolactobacillus Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- PLZWCRUDSJGCLS-UHFFFAOYSA-N calcium;pyridine-2,3-dicarboxylic acid Chemical compound [Ca].OC(=O)C1=CC=CN=C1C(O)=O PLZWCRUDSJGCLS-UHFFFAOYSA-N 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000001136 chorion Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000000266 injurious effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- LVPMIMZXDYBCDF-UHFFFAOYSA-N isocinchomeronic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)N=C1 LVPMIMZXDYBCDF-UHFFFAOYSA-N 0.000 description 1
- -1 isopentenyl Chemical group 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000002015 leaf growth Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229940040461 lipase Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- GJAWHXHKYYXBSV-UHFFFAOYSA-N pyridinedicarboxylic acid Natural products OC(=O)C1=CC=CN=C1C(O)=O GJAWHXHKYYXBSV-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003044 randomized block design Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000002688 soil aggregate Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 229940023877 zeatin Drugs 0.000 description 1
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the biotechnical field, and concretely relates to a peanut rhizosphere biocontrol bacterium, and a preparation method and an application thereof. The peanut rhizosphere biocontrol bacterium is an active strain LX12, the strain LX12 is identified as Brevibacillus laterosporus through 16SrDNA, and the strain is a bacterium screened from peanut rhizosphere soil, antagonistic to fungi, is preserved in China General Microbiological Culture Collection Center, and has a preservation number of CGMCC7420. The strain has an obvious antagonistic effect on pathogens, standoff tests show that the bacterium has a substantial inhibition effect on peanut diseases comprising peanut root rot, southern blight, scab, Phyllosticta glycine and the like, and pot and field tests show that the bacterium has an obvious control effect on the peanut root rot, southern blight, scab, Phyllosticta glycine and the like.
Description
Technical field
The invention belongs to biological technical field, be specifically related to strain peanut rhizosphere biocontrol microorganisms and its preparation method and application.
Background technology
In the research application to biocontrol bacteria, genus bacillus and pseudomonas are to study two kinds of the most deep biocontrol bacterias.Genus bacillus (Bacillaceae) is a section for bacterium, can form bacillus or the coccus of gemma (statospore).Comprise bacillus, Sporolactobacillus, fusobacterium, Desulfotomaculum and gemma Sarcina etc.Injurious factor resistibility is strong to external world for they, distributes wide, is present in soil, water, air and animal intestinal etc. and locates.Gemma is hypopus, and the overwhelming majority is that a thalline only forms a gemma gemma and is positioned at thalline, by core, cortex, spore coat and outer wall, formed, and be gram-positive microorganism.Core is the protoplastis of gemma, includes DNA, RNA, the special gemma protein that may be associated with DNA and synthetic protein and energy-producing system.In addition, also have a large amount of pyridine dicarboxylic acid calcium to be covered with whole gemma.Because gemma has multilayered structure thick and that water content is low, so refractivity is strong, to not easy coloring of dyestuff, gemma has stronger resistibility to hot, dry, radiation, chemostefilant and other chemical factors, and the high-content pyridine dicarboxylic acid that this may be unique with gemma is relevant.
Genus bacillus has a relative superiority in nature soil as microbial population, and the natural isolated strains that many proterties are good is successfully applied to the biological control of Plant diseases.For probing into of the antibacterial product of genus bacillus, find that its antagonistic substance mainly comprises to be mainly the antibiotic that the antibacterial protein of enzyme and secondary metabolism produce, also comprise in addition a part of volatile antimicrobial substance.
It is the most obvious feature that this population of genus bacillus and other populations are distinguished that gemma has heat-resisting resistance.This is conducive to genus bacillus as production, the formulation of biocontrol fungicide and in environment, survives, surely grows and breeding.
As the Brevibacillus laterosporus of bacillus, it mainly contains following characteristics:
The first, can improve soil microenvironment, promote crumb structure to form, the improvement soil texture, increases gram-positive microorganism and Gram-negative bacteria ratio.The ratio increase of gram-positive microorganism and Gram-negative bacteria is the sign that soil quality improves.Effectively the amount reproduction of bacterium produces viscous secretion, increases the soil organism, promotes the formation of soil aggregate, improves permeability, heat retaining property, the nutrient preserving capability of soil.
The second, can stimulate crop root to grow, promote plant growth, improve crop quality.In soil, apply Brevibacillus laterosporus, by its vital movement, secrete various extracellular enzymes as chitinase, proteolytic enzyme, amylase, carbon solution enzyme, hemicellulase, lipase etc. and natural phytohormone (as zeatin, growth hormone, Plant hormones regulators,gibberellins and isopentenyl gland purine etc.), promote the absorption of crop to nutrient, organic matter decomposition abundant in soil, it is the utilizable nutritive substance of plant, various plants hormone, stimulate plant growth and root system development, thus increasing both production and income.Promote leaf growth, increase photosynthetic area and intensity, thereby increase photosynthate synthetic (being that carbon is synthetic), promote to expand root absorbing area by root growth, strengthen improving activity of root system, guarantee synthetic product desired raw material (N, P, K, Mg).
The 3rd, alleviate disease and pest, reduce pesticide residue.When a large amount of beneficial microorganisms is devoted in soil, they are propagation formation dominant group rapidly, and occupy certain space at plant rhizosphere, the nutritive substance that consumption can be utilized by pathogenic bacteria, growth, the breeding of pathogenic bacteria have greatly been weakened, make pathogenic bacteria lose competitive power, this is that probiotics is to pathogenic bacteria " competitive inhibitory effect ".Brevibacillus laterosporus is in vital movement process, and secretion erosion chitinase, fungi, contain a considerable amount of chitins in line eggs wall, and erosion chitinase has decomposed the chitin in wall, makes its protoplastis flow out inactivation, thereby plays the effect of killing disease worm.Ca, the Si, the K that strengthen supply, thicken plant related organization cell walls, and fibrosis, degree of lignification improve, and form the two silicon layers of cutin outward at epidermal area, forms the barrier that stops together germ invasion and attack.Erosion chitinase energy cracking nematode chorion and the fungal cell wall of secretion, life-time service is substantially suitable to nematode, fungal disease prevention effect and chemical pesticide, and diseases prevention, disease-resistant, anti-continuous cropping effect are remarkable.
The 4th, strengthen photosynthesis, improve chemical fertilizer utilization ratio, reduce nitrate content.Use the crop chlorophyll content of Brevibacillus laterosporus generally higher than contrast, improve photosynthesis ability, can the sufficient carbon skeleton of supplying with crop protein synthesis, impel the very fast synthetic protein of nitrogen that absorbs plant materials, promote the conversion of nitrogen, Shaoshi chemical fertilizer, improves cost utilization ratio.The rapid conversion of nitrogen causes nitrate content in plant materials to reduce.
The 5th, solidify some heavy metals, reduce heavy metal content in plant materials.Effectively the vital movement of bacterium changes envrionment conditions, and other elementary states, organic matter etc., activate, solidified some elements.
As the main Biocontrol Mechanism of biocontrol microorganisms, the secreted microbiotic of biocontrol microorganisms has played of paramount importance effect, and Competition and bacteriolysis etc. are also in the effect of bringing into play separately simultaneously.But also there are problems in biocontrol microorganisms in control of plant disease, subject matter is that biocontrol microorganisms does not reach laboratory experiment effect under field condition, while specifically using in field, biocontrol microorganisms is usually subject to the impact of temperature, humidity, PH and agriculture chemical, is in particular in that colonazition is poor, resistance is poor, poor stability.
Brevibacillus laterosporus belongs to bacillus, and Gram-positive can be changed into feminine gender.Peng Qingzhong etc. measure this bacterioid gramstaining result and change along with the variation in the cycle of growing in experiment: when lag period and stationary phase, be Gram-negative, during logarithmic phase, be positive.The bacterial classification of only having got logarithmic phase in experiment is measured, so incomplete to Gram-stained experimental result.Genus bacillus has the features such as breeding is quick, vitality is strong, safety non-toxic; Brevibacillus laterosporus can be secreted synthetic multiple organic acid, enzyme, physiologically active substance etc.
Less at China's report to the inhibition Mechanism Study of plant epiphyte about Brevibacillus laterosporus.Zhao Qiumin etc. have studied bacillus laterosporus the restraining effect of the pathogenic fungies such as gibberella saubinetii (Fusarium graminearum), cotton standing dead bacterium (Rhizoctonia solani), apple wheel line bacterium (Physalospora piricola), aspergillus niger (Aspergillium niger), bread mould (Rhizopus stolonifer), Fusarium oxysporum (Fusarium oxysporum) have been studied in 2006, tentatively determined that antibacterial substance is chitinase.
In recent years, the application of biocontrol bacteria is subject to researchist's attention day by day, its fungistatic effect is also along with progress of research is improved, in occurring in nature screening, obtain antagonism biocontrol microorganisms, and then it is modified or by the change of condition, its fungistatic effect is strengthened by the method for biotechnology, probe into its using method, general is all to make bacteria agent by the method for fermentation, fermentation both can have been produced biocontrol microorganisms, also can produce antibacterial substance by the change of fermentation condition, scientific payoffs is applied in actual production.
Summary of the invention
The problem that the present invention solves is to utilize a kind of wide spectrum that can be used for control of plant disease of experiment field peanut rhizosphere soil resource exploitation, efficient, the active bacterial strain LX12 that can industrialization produces, and cultivation, separation, the evaluation of active bacterial strain LX12 and this bacterial strain, mensuration and the application in control Roots of Peanut maize ear rot, southern blight, shot hole and brown spot of Antifungi ability are provided.
The biomaterial that the present invention joins certificate is LX12 bacterial strain, Classification And Nomenclature is Brevibacillus laterosporus (Brevibacillus laterosporus), the survival after testing on 04 07th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3, Institute of Microorganism, Academia Sinica.
Technical scheme of the present invention is as follows particularly:
One strain peanut rhizosphere biocontrol microorganisms, it is characterized in that, this bacterial strain is active bacterial strain LX12, by 16SrDNA, identify that bacterial strain LX12 is Brevibacillus laterosporus, this bacterial strain is from peanut rhizosphere soil, to filter out the bacterium that fungi is had to Antagonism, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC7420.
Active bacterial strain LX12 of the present invention, through microscopic examination, staining reaction, the experiment of conventional physio-biochemical characteristics and the 16SrDNA sequential analysis of bacterium colony and form, is accredited as Brevibacillus laterosporus, has following characteristics:
(1) morphologic observation shows: bacterial strain LX12 is white in color, and neat in edge has projection, and smooth surface is moistening, opaque.
(2) physiological and biochemical property is measured: during bacterial strain LX12 logarithmic phase, gramstaining is positive, source of nutrition that can be using Citrate trianion as carbon element, there is activity of catalase, methyl red test is positive, can not decompose the macromolecular substance such as gelatin, starch, glucose fermentation experiment is positive, and sucrose, lactose fermentation are negative.
(3) by 16SrDNA, identify that bacterial strain LX12 is Brevibacillus laterosporus, for next step is laid a good foundation to probing into of Brevibacillus laterosporus.
The separation method of one strain peanut rhizosphere biocontrol microorganisms, it is characterized in that, described separation method is: the triangular flask that takes 5g Shandong Peanut Inst. experiment field peanut rhizosphere soil and put into 45ml distilled water, shake for a moment, absorption upper strata liquid 0.1ml joins and contains in 4.5ml sterilized water test tube, make the suspension of 1:100 concentration, then distinguish West 10
-2with 10
-3the each 0.2ml of soil suspension liquid is added drop-wise on the LB solid medium flat board containing Rifampin, is evenly coated with spreading rod, and substratum is inverted in to 28 ℃ of constant incubators cultivates 1-2 days.The setting-out on LB solid medium flat board of an endorsement bacterium colony on picking flat board is inverted in 28 ℃ of constant incubators and cultivates 1-2 days.
The measuring method of one strain peanut rhizosphere biocontrol microorganisms Antifungi ability, is characterized in that, described measuring method comprises the steps:
(1) cultivation of fungi: in aseptic operating platform, peanut pine root fungus, southern blight, shot hole and peanut Cercospora bacteria are seeded in PDA substratum by the square tiles that tweezers are got about 0.5cm*0.5cm, are inverted in 28 ℃ of greenhouses and cultivate 2-3 days.
(2) inoculated bacteria: picking carries out fungi face-off experiment to the best single bacterium colony LX12 of fungi restraining effect.Until fungi, grow to while accounting for culture dish 1/3 size apart from fungi 1-2cm place inoculated bacteria, continue to place 28 degrees Celsius of hot-house culture 2-3 days, observation fungal growth situation.
The application of an above-mentioned strain peanut rhizosphere biocontrol microorganisms in control peanut Roots of Peanut maize ear rot, southern blight, shot hole and peanut Cercospora bacteria.
(1) bacterial strain fermentation liquor preparation: by access LB liquid nutrient medium after bacterial strain activation, be placed in 28 degree shaking table 180r/min concussions and cultivate 3d.
(2) inoculation of peanut pathogenic bacteria: the Roots of Peanut maize ear rot of preservation, southern blight, shot hole and cercospora brown spot of peanut tissue are cleaned up and ground with pulverizer afterwards, mix with sterile soil after filtered through gauze that to make spore quantity be 106/g soil.Bo Ti lower floor is sterile soil, and upper strata is bacterium soil, thickness 5cm left and right.Peanut is directly sowed.
(3) incidence investigation: before sowing,, with the seed dressing of LX12 biocontrol microorganisms fermented liquid, after planting 15d, 30d, 45d utilize LX12 fermentation liquid irrigating root, each processing 10 basins, every basin 3 strains, every basin waters 100 times of fermented liquid 100mL of dilution at every turn.800 times of liquid of 50% derosal and two contrasts of clear water are set.Plant the incidence of 75 days " Invest, Then Investigate " Roots of Peanut maize ear rots, southern blight, shot hole and the cercospora brown spots of peanut.
Beneficial effect of the present invention:
1. Brevibacillus laterosporus of the present invention has good restraining effect to peanut fungal disease and other several plant Common Diseases.
2. Brevibacillus laterosporus good stability cultural method of the present invention is simple;
3. dual test shows, Brevibacillus laterosporus of the present invention has significant restraining effect to peanut diseases such as Roots of Peanut maize ear rot, southern blight, shot hole and brown patch germs, and potted plant and field test shows that Roots of Peanut maize ear rot, southern blight, shot hole and brown spot are had to obvious prevention effect.
Accompanying drawing explanation
Fig. 1: LX12 colony morphology characteristic figure
Fig. 2: the PCR product of the 16srDNA of bacterial strain LX12
Fig. 3: the 16srDNA sequence homology comparison result of the Brevibacillus laterosporus of Brevibacillus in the 16srDNA sequence of active bacterial strain LX12 and GenBank gene pool
Fig. 4: bacterial strain LX12 to its in GenBank database relevant belong to kind build take 16SrDNA sequence as basic phylogenetic tree
Embodiment
1, the separation of active bacterial strain LX12
Take 5g Shandong Peanut Inst. experiment field peanut rhizosphere soil and put into the triangular flask of 45ml distilled water, shake for a moment, absorption upper strata liquid 0.1ml joins and contains in 4.5ml sterilized water test tube, make the suspension of 1:100 concentration, then West 10-2 and the each 0.2ml of 10-3 soil suspension liquid are added drop-wise on the LB solid medium flat board containing Rifampin respectively, evenly be coated with spreading rod, and substratum is inverted in to 28 ℃ of constant incubators cultivates 2 days.The setting-out on LB solid medium flat board of an endorsement bacterium colony on picking flat board is inverted in 28 ℃ of constant incubators and cultivates 2 days.
Wherein, in the setting-out of LB substratum, cultivate after one day, the about 0.3mm of bacterium colony, is white in color, and neat in edge, has projection, and smooth surface is moistening, opaque.As shown in Figure 1.
2, the Physiology and biochemistry of LX12 bacterium is identified
Gramstaining, Citrate trianion utilization test, catalase test, sugar alcohol fermentation test, Starch Hydrolysis test, gelatin liquification test, methyl red test etc. are carried out Physiology and biochemistry evaluation with reference to < < common bacteria system identification handbook > >.
The Physiology and biochemistry qualification result of LX12 bacterial strain is in Table 1, result shows that LX12 thalline shows as Gram-positive, and source of nutrition that can be using Citrate trianion as carbon element, has activity of catalase, methyl red test is positive, and can not decompose the macromolecular substance such as gelatin, starch.Through contrasting document (Fang Zhongda, 1998; Buchanan etc., 1984), the physio-biochemical characteristics of bacterial strain LX12 and Brevibacillus laterosporus are basically identical, tentatively determine that this bacterial strain is Brevibacillus laterosporus.
The physiological and biochemical property of table 1 active bacterial strain LX12
Reference:“+”present?positive?result;“-”present?negative?result.
3, the 16SrRNA molecules of bacterium is identified
3,1 bacterial strain DNA extraction
Choose bacterial isolates fungi to antagonistic activity, 10 identical bacterium colonies of picking do repetition, carry out the extraction of genomic dna, and its extracting method mainly carries out with reference to TIANGEN TIANamp BACTERia DNA Kit test kit specification sheets, and revise slightly, step is as follows:
(1) get inoculum 1mL, the centrifugal 1min of 10000rpm, supernatant exhausts as far as possible;
(2) in bacterial sediment, add 200 μ L damping fluid GA, shake to thalline and thoroughly suspend;
(3) add 4 μ LRNAase(100mg/mL) solution, concussion 15s, room temperature is placed 5min;
(4) Xiang Guanzhong adds 20 μ L Proteinase K solution, mixes;
(5) add 220 μ L damping fluid GB, concussion 15s, places 10min for 70 ℃, brief centrifugal to remove the globule of cap wall;
(6) add 220 μ L dehydrated alcohols, fully concussion mixes 15s, now may occur flocks, brief centrifugal to remove the globule of cap wall;
(7) solution of previous step gained and flocks are all added in an adsorption column GB3 (adsorption column is put into collection tube), the centrifugal 30s of 12000rpm, outwells waste liquid and will in adsorption column CB3, put into collection tube;
(8) to putting into 500 μ L damping fluid GD(preoperation inspections in adsorption column CB3, whether add income dehydrated alcohol), the centrifugal 30s of 12000rpm, outwells waste liquid and will in adsorption column CB3, put into collection tube;
(9) to putting into 700 μ L rinsing liquid PW(preoperation inspections in adsorption column CB3, whether add income dehydrated alcohol), the centrifugal 30s of 12000rpm, outwells waste liquid and will in adsorption column CB3, put into collection tube;
(10) in adsorption column CB3, put into 500 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, outwells waste liquid and will in adsorption column CB3, put into collection tube;
(11) adsorption column CB3 is put back in collection tube, the centrifugal 2min of 12000rpm, outwells waste liquid, adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material;
(12) adsorption column CB3 is proceeded in clean centrifuge tube, to the unsettled dropping in the middle part 50-200 μ L elution buffer TE of adsorption film, room temperature is placed 5min, and the centrifugal 2min of 12000rpm, collects solution in centrifuge tube ,-20 ℃ of preservations.
3,2PCR and sequencing
(1) DNA extracting carries out pcr amplification with reference to the 16srDNABacterial Identification PCR Kit test kit specification sheets of TaKaRa company, wherein forward primer is: 5 '-AGAGTTTGATCATGG CTCAG-3 ', reverse primer is: 3 '-CGCTTACCTTGTTACGACTT-5 '.Pcr amplification condition is as follows: 94 ℃ of denaturation 5min; 94 ℃ of sex change 1min; 53 ℃ of annealing 1min; 72 ℃ are extended 90s; After 72 ℃, extend 5min, totally 30 circulations.PCR product adopts 1% agarose gel electrophoresis to separate, and EB dyeing is placed under 3UVTMTransilluminator (UVP, USA) to be observed.
(2) according to the primer of design in advance and expection amplified fragments size, in conjunction with Marker, object fragment cuts down under ultraviolet lamp, with reclaiming test kit SilicaBeadDNAGelExtractionKit, carries out the recovery of DNA.16srDNA sequencing carries out sequencing by precious biotechnology (Dalian) company limited.By blast program by measure sequence and GenBank(NCBI, website http://blast.ncbi.nlm.nih.gov/Blast.cgi) in sequence comparison, the 16srDNA sequence that then obtains kind close to test strain sequence, genus from GenBank is determined.
The 16srDNA sequence of bacterial strain LX12:
(16sF)GACGAACGGCGGGCGGGCGTGCTATACATGCAGTCGAGCGAGGGTCTTCGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTGAGACTGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGGTTTTGCTTCGCCTGAAGCGAAACGGAAAGATGGCGCAAGCTATCACTTACAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGCTTTCGGGTCGTAAAGTTCTGTTGTTAGGGAAGAAACAGTGCTATTTAAATAAGGTAGCACCTTGACGGTACCTAACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGCTATGTAAGTCTGATGTTAAAGCCCGAGGCTCAACCTCGGTTCGCATTGGAAACTGTGTAGCTTGAGTGCAGGAGAGGAAAGTGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGCCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCAATACCCTTAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGACCTTACCAGGTCTTGACATCCCACTGACCGCTCTAGAGATAGAGCTTCCCTTCGGGGCAGTGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGAATGTTGGATTAGTCCGCACGAGCGGCAACCCTTAATCTTTAGGTGCCAGCATTCAGTTGGGCACTCTTGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACTTCGCGAGAAGATGCTAATCTCTTAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGTAAGGAGCCAGCCGCCGAAGGTGGGGTAGATAACTGGGGTGAAGTCGTAACAA(16sR)
4, the 16srDNA sequential analysis of LX12 bacterium
Take the genomic dna of bacterial strain as template, utilize the 16srDNA Bacterial Identification PCR Kit test kit of TaKaRa company to carry out pcr amplification to the 16srDNA sequence of LX12 bacterial strain, amplification is shown in Fig. 2.The 16srDNA clip size of finding LX12 bacterial strain by electrophoresis detection is about 1400bp, and clip size conforms to test kit desired design.PCR product purification directly carries out sequencing after reclaiming.Measure the total 1467nt base pair composition of 16srDNA fragment that sequence results shows LX12 bacterial strain.Utilize the blast program on NCBI website to carry out sequence alignment to this sequence, obtain the relevant information of the genus and species similarly of having named, comparison result is shown in Fig. 3, result shows the 16srDNA sequence height homology of the Brevibacillus laterosporus (Brevibacillus laterosporus) of Brevibacillus in the 16srDNA sequence of this active bacterial strain and GenBank gene pool, and homology reaches 99%.Build and grow tree and homology analysis result demonstration (Fig. 4), the Brevibacillus laterosporus (Brevibacillus laterosporus) (Accession No:NR037005.1) of bacterial strain LX12 and Brevibacillus forms separately a branch, nearest in evolution, reflect that between them, sibship is nearest, using DANMAN software analysis to obtain the two homology is 99%.In conjunction with traditional physio-biochemical characteristics evaluation and the result of 16SrDNA sequential analysis, judge that bacterial strain LX12 is as Brevibacillus laterosporus.
5, the mensuration of active bacterial strain LX12 Antifungi ability
(1) cultivation of fungi: in aseptic operating platform, peanut pine root fungus and the peanut Cercospora bacteria of getting the fritter of about 0.5cm*0.5cm with tweezers are seeded in PDA substratum is inverted and is cultivated 3 days in 28 ℃ of greenhouses.
(2) inoculated bacteria: single bacterium colony LX12 that picking is best to fungi restraining effect, carries out fungi face-off experiment.Until fungi, grow to while accounting for culture dish 1/3 apart from fungi 2cm place inoculated bacteria, continue to place 28 degrees Celsius of hot-house cultures 3 days, observe fungal growth situation.
Bacteriostasis rate=[(contrast fungal growth radius-processing fungal growth radius)/contrast fungal growth radius] × 100%.
LX12 carries out 3 times (table 2) altogether to the dual test of Roots of Peanut maize ear rot, and restraining effect is obvious, inhibiting rate be minimum be 64.7%, be up to 77.4%.X12 carries out 3 times (table 3) altogether to the dual test of peanut sclerotium rolfsii, and restraining effect is obvious, inhibiting rate be minimum be 66.2%, be up to 70.8%.X12 carries out 3 times (table 4) altogether to the dual test of Peanut Scab, and restraining effect is obvious, inhibiting rate be minimum be 64.3%, be up to 77.5%.LX12 carries out 3 times (table 5) altogether to the dual test of the cercospora brown spot of peanut, and restraining effect is obvious, and inhibiting rate is minimum is 63.3%, is up to 79.8%.Show thus, LX12 has obvious antagonistic action to pathogenic bacteria, can be used as potentiality biocontrol strain.
The face-off experimental result (three batches of experiments, each three repetitions) of table 2LX12 and Roots of Peanut maize ear rot
The face-off experimental result (three batches of experiments, each three repetitions) of table 3LX12 and peanut sclerotium rolfsii
The face-off experimental result (three batches of experiments, each three repetitions) of table 4LX12 and Peanut Scab
The face-off experimental result (three batches of experiments, each three repetitions) of table 5LX12 and the cercospora brown spot of peanut
6, the potted plant and field control peanut disease of bacterial strain LX12 test
(1) bacterial strain fermentation liquor preparation: by access LB liquid nutrient medium after bacterial strain activation, be placed in 28 degree shaking table 180r/min concussions and cultivate 3d.
(2) pot experiment: the Roots of Peanut maize ear rot of preservation, southern blight, shot hole and brown spot tissue are cleaned up and ground with pulverizer afterwards, mix with sterile soil after filtered through gauze that to make spore quantity be 106/g soil.Bo Ti lower floor is sterile soil, and upper strata is bacterium soil, thickness 5cm left and right.Peanut is directly sowed.
(3) field test: experimental field test farm, Shandong Peanut Inst. Laixi carries out, is peanut continuous cropping field, and Roots of Peanut maize ear rot, southern blight, shot hole and brown spot occur serious.Adopt randomized block design, zone leader 4m, wide 4.5m, line-spacing 45cm, spacing in the rows 16.7cm, repeats 3 times, the contrast take 800 times of liquid of 50% derosal as medicament, clear water is blank.In sowing time, after planting 15d, 30d, 45d utilize LX12 fermentation liquid irrigating root, 100 times of about 30mL of fermented liquid of dilution are watered in every strain at every turn respectively.Other field management are produced with normal, 75 days " Invest, Then Investigate " incidences.
Root rot grade scale:
0 grade: on stem foot and main fibrous root all without scab;
1 grade: on stem foot and main root, have a small amount of scab;
3 grades: on stem foot and main root, scab is more, lesion area accounts for 1/4～1/2 of stem foot and the root total area;
5 grades: on stem foot and main root, scab is many and large, and lesion area accounts for 1/2～3/4 of stem foot and the root total area;
7 grades: on stem foot and main root, scab in flakes, forms around stem phenomenon, but root system is not dead;
9 grades: root system necrosis, plant overground part is wilted or be dead.
Southern blight grade scale:
0 grade: plant is asymptomatic;
1 grade: only at basal part of stem, produce scab;
2 grades: basal part of stem produces the contracting symptom of hanging, the existing systemic symptom of 1/3rd following tables of whole strain (withered, death, wilting etc.);
3 grades: the systemic symptom of performance below 2/3rds of whole strain;
4 grades: 2/3rds above representation system symptoms of whole strain.
Shot hole grade scale:
0 grade: healthy plant
1 grade: on top tender leaf and carpopodium, occur little scab
2 grades: on tender leaf, carpopodium, stem, occur little scab
3 grades: tender leaf edge upsweeps, on peanut stem and carpopodium, there is scab shape
4 grades: carpopodium, stem are seriously bending, the existing calcination shape of plant
Brown spot grade scale:
0 grade: disease-free symptom;
1 grade: the blade area of being injured accounts for below 1/10 of blade area of investigation;
2 grades: the blade area of being injured accounts for below 1/4 of blade area of investigation;
3 grades: the blade area of being injured accounts for below 1/2 of blade area of investigation;
4 grades: the blade area of being injured accounts for investigation the more than 1/2 of blade area, fallen leaves.
Diseased plant rate=morbidity strain number/total strain number × 100%
Disease index=∑ (morbidity level typical value × diseased plant numbers at different levels) × 100/ (investigating total strain number × superlative degree morbidity typical value)
Prevention effect=[(contrast disease index-processing disease index)/contrast disease index] × 100%
With the fermentation liquor treatment peanut bacterial strain of biocontrol microorganisms LX12, indoor pot and field experiment all show that Roots of Peanut maize ear rot, southern blight, shot hole and brown spot are all had to obvious prevention effect, LX12 is 51.3% to the prevention effect of indoor pot Roots of Peanut maize ear rot, and field control effect is 54.6%(table 6); LX12 is 58.9% to the prevention effect of indoor pot peanut sclerotium rolfsii, and field control effect is 54.9%(table 7); LX12 is 51.4% to the prevention effect of indoor pot Peanut Scab, and field control effect is 57.4%(table 8); LX12 is 60.9% to the prevention effect of the indoor pot cercospora brown spot of peanut, and field control effect is 52.4%(table 9), all higher than the processing of contrast medicament derosal.
The prevention effect of table 6 bacterial strain LX12 to Roots of Peanut maize ear rot
The prevention effect of table 7 bacterial strain LX12 to peanut sclerotium rolfsii
The prevention effect of table 8 bacterial strain LX12 to Peanut Scab
The prevention effect of table 9 bacterial strain LX12 to the cercospora brown spot of peanut
Claims (5)
1. a strain peanut rhizosphere biocontrol microorganisms, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is characterized in that, this bacterial strain is active bacterial strain LX12, and deposit number is CGMCC 7420.
2. a microbiobacterial agent prepared by the peanut rhizosphere biocontrol microorganisms that is CGMCC 7420 by deposit number described in claim 1.
3. a preparation method for peanut rhizosphere biocontrol microorganisms as claimed in claim 1, is characterized in that, comprises following steps:
Step 1) takes 5g peanut rhizosphere soil puts into the triangular flask of 45ml distilled water, shake a moment, draws upper strata liquid 0.1ml and joins and contain in 4.5ml sterilized water test tube, makes the suspension of 1:100 concentration;
Step 2) so draw respectively 10
-2with 10
-3the each 0.2ml of soil suspension liquid is added drop-wise on the LB solid medium flat board containing Rifampin, is evenly coated with spreading rod, and substratum is inverted in to 28 ℃ of constant incubators cultivates 1-2 days;
The setting-out on LB solid medium flat board of an endorsement bacterium colony on step 3) picking flat board is inverted in 28 ℃ of constant incubators and cultivates 1-2 days.
4. a measuring method for peanut rhizosphere biocontrol microorganisms Antifungi ability as claimed in claim 1, is characterized in that, comprises following steps:
The cultivation of step 1) fungi: in aseptic operating platform, peanut pine root fungus, southern blight, shot hole and the peanut Cercospora bacteria of getting respectively 0.5cm*0.5cm fritter are seeded in PDA substratum is inverted and is cultivated 2-3 days in 28 ℃ of greenhouses;
Step 2) inoculated bacteria: picking carries out fungi face-off experiment to the best single bacterium colony LX12 of fungi restraining effect; Until fungi, grow to while accounting for culture dish 1/3, apart from fungi 1-2cm place inoculated bacteria, continue to place in 28 ℃ of greenhouses and cultivate 2-3 days.
5. a peanut rhizosphere biocontrol microorganisms as claimed in claim 1 is in the application preventing and treating in Roots of Peanut maize ear rot, southern blight, shot hole and peanut Cercospora bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310643869.3A CN103756930B (en) | 2013-12-03 | 2013-12-03 | Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310643869.3A CN103756930B (en) | 2013-12-03 | 2013-12-03 | Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103756930A true CN103756930A (en) | 2014-04-30 |
| CN103756930B CN103756930B (en) | 2015-06-10 |
Family
ID=50524257
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310643869.3A Expired - Fee Related CN103756930B (en) | 2013-12-03 | 2013-12-03 | Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103756930B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480046A (en) * | 2014-12-18 | 2015-04-01 | 江苏省农业科学院 | Brevibacillus laterosporus strain and application thereof |
| CN104673730A (en) * | 2015-03-23 | 2015-06-03 | 大连理工大学 | Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof |
| CN107135828A (en) * | 2017-04-30 | 2017-09-08 | 中国农业科学院油料作物研究所 | A kind of peanut sclerotium rolfsii greenhouse Seedling Inoculation method |
| CN108048354A (en) * | 2017-12-26 | 2018-05-18 | 山东省花生研究所 | One bacillus subtilis and its application |
| CN108102972A (en) * | 2018-01-31 | 2018-06-01 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | A kind of preparation method and application of the compound biocontrol fungicide of cure plant disease of peanut |
| CN111849815A (en) * | 2020-07-21 | 2020-10-30 | 广西民族大学 | Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion |
| CN111944728A (en) * | 2020-08-26 | 2020-11-17 | 河南大学 | Pseudomonas chlororaphis and application thereof |
| CN113248329A (en) * | 2021-07-02 | 2021-08-13 | 山东丰本生物科技股份有限公司 | Microbial fertilizer with continuous cropping resistance effect and preparation method and application thereof |
| CN116162564A (en) * | 2022-08-18 | 2023-05-26 | 西南科技大学 | Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580242A (en) * | 2004-05-18 | 2005-02-16 | 北京生太平生物工程技术有限公司 | Laterosporo short bacillus, and its fermentating process and use |
| CN1631166A (en) * | 2004-11-04 | 2005-06-29 | 云南大学 | Method for preparation biological bacterium for preventing and curing pseudo-ginseng root rot and its application |
| CN102838398A (en) * | 2011-09-05 | 2012-12-26 | 麻林涛 | Special compound microbial fertilizer for peanuts and method for preparing special compound microbial fertilizer |
-
2013
- 2013-12-03 CN CN201310643869.3A patent/CN103756930B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580242A (en) * | 2004-05-18 | 2005-02-16 | 北京生太平生物工程技术有限公司 | Laterosporo short bacillus, and its fermentating process and use |
| CN1631166A (en) * | 2004-11-04 | 2005-06-29 | 云南大学 | Method for preparation biological bacterium for preventing and curing pseudo-ginseng root rot and its application |
| CN102838398A (en) * | 2011-09-05 | 2012-12-26 | 麻林涛 | Special compound microbial fertilizer for peanuts and method for preparing special compound microbial fertilizer |
Non-Patent Citations (3)
| Title |
|---|
| SHOJI J ET AL.: "Isolation of a new antibiotic, laterosporamine. Studies on antibiotics from the genus Bacillus.XIII", 《ANTIBIOT (TOKYO)》, 31 December 1976 (1976-12-31), pages 390 - 393 * |
| 周峻沛等: "利用Brevibacillus laterosporusYMF3.00003菌株研制生物杀菌剂", 《云南大学学报(自然科学版)》, 31 December 2006 (2006-12-31), pages 456 - 460 * |
| 柯崇榕等: "侧孢短芽胞杆菌产生线虫侵染性蛋白酶的优化", 《微生物学杂志》, 30 November 2010 (2010-11-30), pages 6 - 10 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480046A (en) * | 2014-12-18 | 2015-04-01 | 江苏省农业科学院 | Brevibacillus laterosporus strain and application thereof |
| CN104480046B (en) * | 2014-12-18 | 2017-04-05 | 江苏省农业科学院 | One plant of Brevibacillus laterosporus and its application |
| CN104673730A (en) * | 2015-03-23 | 2015-06-03 | 大连理工大学 | Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof |
| CN107135828B (en) * | 2017-04-30 | 2019-10-29 | 中国农业科学院油料作物研究所 | A kind of peanut sclerotium rolfsii greenhouse Seedling Inoculation method |
| CN107135828A (en) * | 2017-04-30 | 2017-09-08 | 中国农业科学院油料作物研究所 | A kind of peanut sclerotium rolfsii greenhouse Seedling Inoculation method |
| CN108048354A (en) * | 2017-12-26 | 2018-05-18 | 山东省花生研究所 | One bacillus subtilis and its application |
| CN108048354B (en) * | 2017-12-26 | 2020-06-16 | 山东省花生研究所 | Bacillus subtilis and application thereof |
| CN108102972A (en) * | 2018-01-31 | 2018-06-01 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | A kind of preparation method and application of the compound biocontrol fungicide of cure plant disease of peanut |
| CN111849815A (en) * | 2020-07-21 | 2020-10-30 | 广西民族大学 | Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion |
| CN111849815B (en) * | 2020-07-21 | 2022-11-18 | 广西民族大学 | Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion |
| CN111944728A (en) * | 2020-08-26 | 2020-11-17 | 河南大学 | Pseudomonas chlororaphis and application thereof |
| CN111944728B (en) * | 2020-08-26 | 2022-02-22 | 河南大学 | Pseudomonas chlororaphis and application thereof |
| CN113248329A (en) * | 2021-07-02 | 2021-08-13 | 山东丰本生物科技股份有限公司 | Microbial fertilizer with continuous cropping resistance effect and preparation method and application thereof |
| CN116162564A (en) * | 2022-08-18 | 2023-05-26 | 西南科技大学 | Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof |
| CN116162564B (en) * | 2022-08-18 | 2023-12-01 | 西南科技大学 | Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103756930B (en) | 2015-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103756930B (en) | Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof | |
| CN103146586B (en) | Trichoderma harzianum strain for controlling plant fungus diseases and application thereof | |
| CN101974438B (en) | Eucalyptus endophyte and application thereof | |
| CN105567615A (en) | Bradyrhizobium sp. and application thereof | |
| CN103931664B (en) | A kind of complex micro organism fungicide that adopts is used for the cryophylactic method of Festuca Arundinacea | |
| CN107646616A (en) | A kind of preparation method of floriculture substrate | |
| CN105543132A (en) | Bacillus methylotrophicus YB-F7 and application thereof in preventing plant diseases | |
| CN112746024A (en) | Trichoderma composite microbial agent and bio-organic fertilizer prepared from same | |
| CN108048354A (en) | One bacillus subtilis and its application | |
| CN101519644B (en) | Sinorhizobium sp. and application thereof | |
| CN109182219A (en) | One plant of Mo Haiwei bacillus for promoting the growth of Henry David Thoreau grass and its application | |
| CN109971656B (en) | Ginger endogenetic trichoderma viride and application thereof | |
| CN105439657B (en) | A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry | |
| CN106222121B (en) | A kind of bacillus megaterium bacterial strain, biocontrol agent and the preparation method and application thereof | |
| CN101748088B (en) | Bacterial strain of root nodule nitrogen-fixing strain series RY3 and application thereof | |
| CN100575476C (en) | The bacterial strain B 211 of control cotton verticillium wilt | |
| CN108102972B (en) | Preparation method and application of composite biocontrol microbial inoculum for preventing and treating peanut diseases | |
| CN110343621A (en) | A kind of Trichoderma asperellum strain and its application | |
| CN106035378A (en) | Compound bacterium agent for preventing and treating bacterial diseases of tobacco | |
| CN105316258B (en) | It is a kind of prevent and treat rice sheath blight disease bacterial strain 1LN2 and its application | |
| CN102533564B (en) | A kind of screening method of bio-control trichoderma in corn seedling stage root rot period | |
| CN110205249B (en) | Method for promoting plant growth and alternaria alternate fungus used by same | |
| CN107586748B (en) | A kind of China's sporangium and its application | |
| CN103602622B (en) | Rhodopseudomonas bacterial strain and the application in suppression bacterium of downy mildew of cucumber, promotion cucumber growth thereof | |
| CN104593266B (en) | A kind of Endophytic Fungi in Tomato intertexture branch top spore and its application in tomato root-knot eelworm diease occurrence is anti- |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANDONG PEANUT INST. Free format text: FORMER OWNER: QINGDAO RUNDIFENG TECHNOLOGY CO., LTD. Effective date: 20150511 Owner name: QINGDAO RUNDIFENG TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHANDONG PEANUT INST. Effective date: 20150511 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 266071 QINGDAO, SHANDONG PROVINCE TO: 266000 QINGDAO, SHANDONG PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20150511 Address after: 266000 Shandong province Qingdao City, Lianyungang Road No. 20 Applicant after: Shandong Peanut Inst. Applicant after: Qingdao Rundifeng Technology Co.,Ltd. Address before: 266071 Nanning Road, Wangcheng Industrial Park, Qingdao, Shandong, Laixi Applicant before: Qingdao Rundifeng Technology Co.,Ltd. Applicant before: Shandong Peanut Inst. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170605 Address after: Wangcheng Street office Linquan village 266000 Shandong province Qingdao city of Laixi Patentee after: Qingdao Xiangrun Biotechnology Co., Ltd. Address before: 266000 Shandong province Qingdao City, Lianyungang Road No. 20 Co-patentee before: Qingdao Rundifeng Technology Co.,Ltd. Patentee before: Shandong Peanut Inst. |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150610 Termination date: 20181203 |